Seven New Species of the Genus Geastrum (Geastrales, Geastraceae) in China

Geastrum belongs to Basidiomycota, Agaricomycetes, Geastrales, and Geastraceae. The genus Geastrum exoperidium normally splits at maturity into a characteristic star-like structure. It is a saprophytic fungus with great research significance. Based on morphological observation combined with phylogenetic analysis through ITS and LSU, seven new species of Geastrum belong to four sections, viz., Sect. Myceliostroma, Geastrum laneum; Sect. Exareolata, Geastrum litchi, Geastrum mongolicum; Sect. Corollina, Geastrum pseudosaccatum, Geastrum melanorhynchum, Geastrum oxysepalum; and Sect. Campestria, Geastrum microphole. Illustrated descriptions and the ecological habits of the novel species are provided.


Introduction
The genus Geastrum is a type of gasteroid Basidiomycota, which has been recorded on all continents except Antarctica, mostly in the forest humus layer, although it is occasionally seen on rotten wood or sand and grassland [1]. Some species of Geastrum are medicinal fungi with vital research value, and some species are widely used in forestry production practice, as they are able to enhance the absorption function of forest roots and improve the survival rate of afforestation [2]. They can be used as a natural hygrometer based on whether the exoperidium is hygroscopic [2]. As of November 2022, the Index Fungorum recorded more than 100 species.
However, there is still a lot to be studied and explored in the richness of the species resources of the Geastrum in China. In our recent investigations on the Geastrum from China over the past two years, seven new species were found. They were described in detail and illustrated.

Morphological Study
Dried specimens used in this study were deposited at the Herbarium of Mycology of Jilin Agricultural University (HMJAU), China. The methodology and notation used here followed those of Cai et al. (2016) and Cui et al. (2018) [30,31].
Macromorphological descriptions were based on fresh specimens, which were photographed in the field with notes and laboratory supplemental measurements. The color description of the basidiomata was based on Kornerup and Wanscher (1978) [32]. Micromorphological studies were carried out using a light microscope and scanning electron microscope. Dried specimens were used to observe microscopic features. Data of the sections (basidiospores, basidia, capillitial hyphae, and exoperidium) were obtained from dried specimens, which were rehydrated in 5% KOH or stained in Congo red when necessary, and the light microscope (Olympus BX50) was used for the examination of microscopic structures with a high-resolution oil objective lens (1000×). The dimensions of basidiospores are given using a notation in the form 'a-b'. The dimensions of basidium are given using a notation in the form 'c-d × e-f'. The dimensions of capillitial hyphae are given using a notation in the form 'g-h'. The abbreviation [n/m/p] represents n basidiospores measured from m basidiomata of p collections. The range 'a-b' means the minimum to the maximum of the diameter. The range 'c-d' means the minimum to maximum length, 'e-f' and'g-h' means the minimum to maximum width.
For scanning electron microscopy, air-dried samples were mounted on a sample holder covered with double-sided adhesive tape, sprayed with pure gold until fully coated using an ion sputtering instrument IXRF MSP-2S, and observed with a Hitachi SU8010.

DNA Extraction, Amplification and Sequencing
Genomic DNA was extracted from 0.1 to 0.2 mg of dried specimen using a NuClean Plant Genomic DNA kit (CWBIO, Beijing, China) and preserved at −20 • C. The 30 µL PCR reaction system is shown in Table 1. Two molecular markers were investigated, i.e., ITS1F (3 -CTTGGTCATTTAGAGGAAGTAA-5 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ), which were used as primers for the internal transcribed spacer (ITS) (White et al. 1990, Gardes et al. 1993) [33,34]. LR0R (5 -ACCCGCTGAACTTAAGC-3 ) and LR5 (5 -ATCCTGA GGGAAACTTC-3 ) were used for the large subunit of the nuclear ribosomal RNA gene (nrLSU). The PCR procedure for ITS (including 5.8 S) was as follows: initial denaturation at 94 • C for 4 min, followed by 35 cycles at 94 • C for 35 s, 54 • C for 35 s, and 72 • C for 45 s, and a final extension of 72 • C for 10 min. The PCR procedure for nrLSU was as follows: initial denaturation at 94 • C for 4 min, followed by 35 cycles at 94 • C for 1 min, 53 • C for 1 min, 72 • C for 1 min, and a final extension of 72 • C for 10 min. The PCR products were purified and sequenced in Bioengineering (Shanghai) Co., Ltd., China, with the same primers. The newly generated sequences were deposited at GenBank (https://www.ncbi.nlm.nih.gov (accessed on 8 December 2022)). All sequences analyzed in this study were deposited at GenBank and are listed in Table 2.

Phylogenetic Analyses
The new sequences generated in this study were combined with the sequences downloaded from GenBank and outgroups Schenella pityophila  [11,35]. Detailed information for these sequences is given in Table 1. After PCR amplification, unidirectional sequencing of ITS products followed. Then, the products of nrLSU were sequenced in a bidirectional sequence and were assembled using a Sequencher 5.4.5 (Gene Codes, Ann Arbor, Michigan, USA). DNA sequences were aligned using MAFFT 7.110 with the G-INI-I option, while the few ambiguously aligned regions of the ITS and nrLSU alignments were removed with Gblocks v.0.91b, by keeping the default settings but allowing all gap positions when not ambiguous and manually adjusted in Sequencher 5.4.5 [36,37].
Maximum likelihood (ML) analysis was performed in RAxML v8.2.4 with GTRGAMMA model [38]. The best tree was obtained by executing 100 rapid bootstrap inferences, and thereafter a thorough search for the most likely tree using one distinct model/data partition with joint branch length optimization. ModelFinder was used to select the best-fit partition model (Edge-linked) using Bayesian information criterion (BIC) [39]. Best-fit model, according to BIC, were the GTR+I+G+F model for the ITS subset (1-620) and the GTR+I+G+F model for the nrLSU subset (621-1584). We used the GTR+I+G+F model. Bayesian Inference phylogenies were inferred using MrBayes 3.2.6; under partition model (two parallel runs, 10,554,300 generations), in which the initial 25% of sampled data were discarded as burn-in, four chains and sampling for every 100th generation four Markov chains (MCMC) were run until the split deviation frequency value was <0.01 [40]. Finally, FigTree version 1.4.3 was used to visualize the phylogenetic trees [41]. Branches that received bootstrap values for maximum likelihood (ML) ≥ 75% and Bayesian inference (BI) ≥ 0.95 were considered as significantly supported.

Phylogeny
A total of 28 new sequences were generated for this study and with the 144 sequences downloaded from GenBank. In the phylogenetic analysis of the combined dataset (ITS, nrLSU), the aligned lengths of the two gene loci were 606 and 962 base pairs. Bayesian and ML analysis resulted in a same topology. Bayesian analysis has an average standard deviation of split frequencies equal to 0.007893. Only the ML tree is provided in Figure 1; ML bootstrap values (≥75%) and PP (≥0.75) are shown at the nodes.
The constructed phylogenetic tree is similar to the branching structure given in . The difference is that the position of the sections in the phylogenetic tree is slightly different. The species in the sections are the same. This phylogenetic tree is different from the branch support rate of the phylogenetic tree established by Zamora. However, these differences are allowed to exist and do not affect the position of the species on the phylogenetic tree.  The phylogenetic tree shows seven new species in four sections. In Sect. Myceliostroma, a new species G. laneum, with high support values for all specimens, was found (PP = 1.00, MLbs = 99%) and formed a sister branch with a higher support value (PP = 0.95) with G. neoamericanum J.O. Sousa, Accioly, M.P. Martín & Baseia. At the same time, this new species and other species in the section can also be well distinguished on the phylogenetic tree.
There are two new species in Sect. Exareolata. One is G. litchi with high support values for all specimens in this section (PP = 1.00, MLbs = 95%) and formed a sister branch with G. argentinum Speg. with a higher support value (PP = 0.99, MLbs = 82%). There is a Long Branch Attraction in the genus of Geastrum. There are also intraspecific variation in G. litchi. Therefore, the two specimens representing G. litchi have a relatively long phylogenetic distance compared with other species in this section. The other is G. mongolicum with high support values for all specimens in this section (PP = 1.00, MLbs = 100%) and with G. rufescens Pers. formed a sister branch with a higher support value (PP = 0.98, MLbs = 64%). These two new species and other species in the section can also be well distinguished on the phylogenetic tree.
There are three new species in Sect. Corollina. The first is G. pseudosaccatum with high support values for all specimens in this section (PP = 1.00, MLbs = 99%) and formed a sister branch with a higher support value (PP = 1.00, MLbs = 89%) with G. saccatum Fr. The second is G. melanorhynchum with high support values for all specimens in this section (PP = 1.00, MLbs = 100%), and the third is G. oxysepalum with high support values for all specimens in this section (PP = 1.00, MLbs= 100%). The last two new species are sister taxa to each other and form a clade with higher support values (PP = 0.78). The last two new species with G. lageniforme Vittad formed a sister branch with a higher support value (PP = 0.99, MLbs = 84%). These three new species and other species in this section can also be well distinguished on the phylogenetic tree.
In Sect. Campestria, a new species G. microphole with high support values for all specimens in this section (PP = 1.00, MLbs = 100%) and with G. pseudostriatum Hollós formed a sister branch with a higher support value (PP = 1.00, MLbs = 100%). At the same time, this new species and other species in this section can also be well distinguished on the phylogenetic tree.  divided the genus into 14 branches based on morphological, chemical, and molecular phylogenetic data, and explained in detail the relationship between the branches they found; all of them were strongly to moderately supported when the results from the three different phylogenetic analyses were combined [11]. This paper mainly uses molecular data to construct a phylogenetic tree, showing the division of 14 parts as shown in ( Figure 1). As noted above, although tree topologies were almost identical between the ML and Bayesian trees, they are different in support rate, which may be related to their respective calculation methods. The low branch support rate may be due to the long branch attraction effect. Because of the lack of other species that have not been found on the branch, the branch length and support rate are affected; this is also the reason why some sections are not very stable, and some sections will have multiple sources. These problems need to be studied through a large number of field collections in the future. However, these differences do not affect the overall branching stability, nor do they affect the branching stability of new species (Figure 1).

Taxonomy
Geastrum laneum T. Bau & X. Wang, sp. nov. (Figures 2 and 3) MycoBank no: MB846867 Diagnosis: Differs from G. mirabile (Mont.) E., Fisch, in terms of the mycelial tufts, the latter expanded basidiomata has a mycoderm at the base, the mycelial layer is not encrusted with debris; peristome fibrillose; basidiospores displays a delicately warry or columnar process [26]  Etymology: 'laneum' refers to its mycelial layer visible coarse short villus in a felted form.
Type Etymology-'litchi' refers to the mycelial layer surface covered with reddish-brown small pyramidal tufts of villus that produce an areolate pattern similar to the surface of a lychee fruit.
Description: Unexpanded basidiomata dark reddish brown (8E6), 0.9-2.3 cm in diameter, white mycelial tufts, and scent of light chocolate. Expanded basidiomata small to medium sized, 1.6-2.4 cm in diameter. Exoperidium: shallowly to deeply saccate, splits into 5-7 lobes at maturity, lobes 0.5-1.1 cm wide, tapered at the front end, rays nonhygroscopic. Pseudoparenchymatous layer: smooth surface, brownish grey (8C2), contracted along margin of lobes or at base of lobes breaking, easily exfoliation, aseptic collar. Fibrous layer: grey (8B1), tightly attached to the mycelial layer. Mycelial layer: surface covered with small reddish brown (8E6) pyramidal tufts of villus that produce an areolate pattern similar to the surface of a lychee fruit, not easily dislodged, not encrusted with debris.   Endoperidial body: globular, 1.2-1.4 cm in diameter, projecting apically or extending into a beak, 0.1-0.2 cm length, sessile, without an apophysis. Endoperidium: brownish grey (8D2) with pale powder, with a smooth surface and greyish villus visible under the dissecting microscope. Peristome: broad-conical, silky fibrillose, shallower or darker in color than the endoperidium, undelimited.
Description: Expanded basidiomata are mostly small to medium sized, 1.2-3.2 cm. Exoperidium: shallowly saccate, arched, dehiscence often greater than halfway down, at maturity splits into 7-9 lobes, lobes 0.4-1.3 cm wide, lobes long and mostly rolled outward to under the outer exoperidial disc, extremely narrow at the apex. Pseudoparenchymatous layer: smooth surface, reddish grey (9B2) or brownish grey (9B3), contracted along margin of lobes or falling off at base of lobes without breaking, aseptic collar. Fibrous layer: white (9A1), tightly attached to the mycelial layer. Mycelial layer: reddish brown (8E7), felt surface, not easily dislodged, not encrusted with debris.
Etymology: 'oxysepalum ' means pointed sepals, also known as acute sepals, and refers to its exoperidium after drying extremely narrow at the apex.
Basidiospores: spherical, 2.7-3.9 µm in diameter, tan in contact with 5% KOH solution, surface with delicately echinulate, length 0.2-0.9 µm, non-starchy, columnar process under scanning electron microscope. Capillitial hyphae up to 1.0-7.0 µm in diameter, thick-walled, brownish yellow, unbranched, wall surface rough, with surface debris. Exoperidium: Distribution: Jilin Province, China. Geastrum microphole T. Bau & X. Wang, sp.nov. (Figures 14 and 15). MycoBank no: MB846873. Diagnosis: Differs from G. berkeleyi Massee by the peristome, which is sulcate, pseudoparenchymatous layer white, pale brownish to gloom chestnut, a few forming collars at base of stalk [25]. Differs from G. pseudostriatum Hollós by the pseudoparenchymatous layer, which is initially greyish pink to pale brownish and later beige-brown to dark brown, peristome sulcate, endoperidium and pseudoparenchymatous layer surface are attached with white frost or white particles [12,13]. Differs from G. pectinatum Pers. by the stalk, which has a base with a long stalk, usually over 3.0 mm length, the pseudoparenchymatous layer displays complete retention or partial shedding, with shedding often forming a collar at the base of the stem [26].
Type  (Figures 14 and 15) MycoBank no: MB846873 Diagnosis: Differs from G. berkeleyi Massee by the peristome, which is sulcate, pseudoparenchymatous layer white, pale brownish to gloom chestnut, a few forming collars at base of stalk [25]. Differs from G. pseudostriatum Hollós by the pseudoparenchymatous layer, which is initially greyish pink to pale brownish and later beige-brown to dark brown, peristome sulcate, endoperidium and pseudoparenchymatous layer surface are attached with white frost or white particles [12,13]. Differs from G. pectinatum Pers. by the stalk, which has a base with a long stalk, usually over 3.0 mm length, the pseudoparenchymatous layer displays complete retention or partial shedding, with shedding often forming a collar at the base of the stem [26].

Discussion
The phylogenetic placement of the Geastrum clades has been discussed by Zamora et al., who found 14 clades within Geastrum [11] The new species are distributed in four sections, viz., Sect. Myceliostroma, Sect. Exareolata, Sect. Corollina, and Sect. Campestria.
Geastrum laneum was clustered with G. neoamericanum in our phylogenetic analyses, and can be distinguished through the presence of an encrustation of debris [43]. Morphologically, G. laneum resembles G. mirabile Mont; they can be distinguished based on differences in the peristome, basidiospore, and whether an encrustation of debris is extant [26]. It differs from G. laevisporum J.O. Sousa & Baseia by the mycelial layer, the latter being orange white, densely intermixed with sediments, felted, peeling away in irregular patches with age exposing the fibrous layer, not persistently [44]. It also differs from G. javanicum Lév. by the mycelial layer and habitat, the latter encrusted with debris and grown in mixed forest or on sandy soil, as well as a few on stumps; peristome fibrillose [26].
Geastrum litchi was clustered with G. argentinum in our phylogenetic analyses, and can be distinguished based on whether they have stalks, differences in the peristome, and Basidiospores: spherical, 3.7-5.0 µm in diameter, yellowish brown to dark brown in contact with 5% KOH solution, surface with a delicately warry or short columnar process, length 0.6-0.9 µm, non-starchy, columnar process under scanning electron microscope. Capillitial hyphae up to 1.0-5.0 µm in diameter, thick-walled, brownish-yellow, unbranched, with sparse surface debris. Exoperidium 557.1-660.4µm thick, the pseudoparenchymatous layer formed of the pseudoparenchymatous of a structured angular cell, 5.3-29.2 × 4.1-28.2 µm; fibrous layer formed of thin-walled interlacing filament tissue, 3.1-5.8 µm; the mycelium layer formed of thin-walled hyphae diamater tissue 1. four species can be readily distinguished by the different texture of the surface of the unexpanded basidiomatas and the structure of the peristome. The G. morganii pseudoparenchymatous layer frequently forms a collar, peristome is undelimited, and is irregularly sulcate. Geastrum reticulatum has the characteristic reticulated pattern created by lines of raised hyphae. The G. triplex with collar and is usually distinctly delimited by a circle of lighter color.
Geastrum oxysepalum was clustered with G. melanorhynchum in our phylogenetic analyses. Morphologically, the two species can be distinguished based on differences in the peristome and whether felt is present on the mycelial layer surface. Geastrum oxysepalum is similar in size and has morphology to G. velutinum Morgan and G. triplex [45,47]. The mycelium layer is often separated from the fiber layer to form two layers of lobes and peristome is not delimited in G. velutinum. Geastrum triplex is a widely distributed species with collar and usually distinctly delimited by a circle of lighter color.
Geastrum microphole was clustered with G. pseudostriatum and G. berkeleyi Massee in our phylogenetic analyses. Morphologically, the three species can be distinguished based on differences in the peristome and whether a crystal is present on the endoperidium surface. Geastrum microphole is similar to G. campestre Morgan and G. pectinatum Pers. [12,26]. All are large species and have stalks. The three species can be readily distinguished by the different texture or color of the surface of the pseudoparenchymatous layer, the structure of the peristome and different from the length of the handle. Pseudoparenchymatous layer is found in young pinkish specimens with age brown to grey brown, greyish fibrous layer, grey to grey brown endoperidium, and is distinctly warty in G. campestre. The stalk of the G. pectinatum is mostly flat and long, 0.3-0.7 cm in diameter.
In this study, through the combination of morphology and molecular data, seven new species of the genus were found in China. It shows that the diversity of forest macrofungi in China is extremely rich (Dai et al. 2021) [48], and it also provides important data, thus supporting the systematic study of the genus in the future. However, there are still many species of Geastrum that lack molecular data, which limits the systematic study of this genus [11]. For the time being, the best gene marker for the identification of most Geastrum species is ITS, while more terminal nodes in phylogenetic trees need to be investigated by utilizing more gene markers, such as tef1 and RPB1. There are only five tef1 sequences and fifteen RPB1 sequence of Geastrum in NCBI (https://www.ncbi.nlm.nih.gov/protein (accessed on 26 November 2022)). It is necessary to obtain more gene fragments to build a more objective phylogenetictree, and therefore, more research needs be carried out in the future.